Normal cells control their cholesterol content within narrow limits. If cholesterol uptake increases, cholesterol efflux is also increased to keep cell cholesterol content the same. If this response is inhibited, free and esterified cholesterol accumulate in the cell. Foam cell formation reflects a failure of this regulatory pathway. The molecular basis of this reflexive cholesterol unloading and its regulation will be determined. We recently observed that caveolae, which are cholesterol-rich cell surface microdomains, are the terminus of the cholesterol efflux pathway to high density lipoprotein. The presence of caveolae at the cell surface is recognized to depend on caveolin, a 22kDa cholesterol-binding protein. Four Specific Aims deal with different aspects of the molecular and cell biology of caveolae related to cholesterol transport and efflux. In the first Specific Aim, the mechanism of induction of caveolin mRNA in response to cholesterol loading by low density lipoprotein will be determined. In the second Specific Aim, human lymphoid cells which lack endogenous caveolin will be stably transfected with human caveolin cDNA. Its effects on the ability of these cells to regulate their cholesterol content will be determined. In the third Specific Aim, the intracellular lipid vesicles that carry cholesterol to the caveolae will be fully characterized, as will the cytoplasmic factors required for cholesterol transport. In the last Specific Aim, the identity of a factor, possibly related to cellubrevin, which targets intracellular cholesterol to the cell surface caveolae, will be established. These studies will provide novel basic information on the biology of cellular cholesterol homeostasis. They will also identify new targets for intervention in promoting the unloading of cholesterol-filled cells.